This grant evaluates hypothalamic accumulation of the sphingolipids ceramide or glucosylceramide can drive attenuation of hypothalamic leptin, insulin, or serotonin action in POMC and AgRP neurons. Overaccumulation of sphingolipids can drive insulin resistance in a number of peripheral tissues. AgRP and POMC neurons within the hypothalamus are two distinct subpopulations of neurons that are known to regulate both body weight and glucose homeostasis in response to insulin or leptin. The hypothesis that POMC neurons accumulate ceramide and glucosylceramides in the obese state, and these sphingolipids drive resistance of these cells to leptin and insulin's glucoregulatory signals will e investigated. To test this, triggered overexpression of an inducible enzyme which degrades ceramides, acid ceramidase, will be promoted in POMC or AgRP neurons of adult mice. This will be complemented with similar mice which prevent glucosylation of ceramide via gain or loss of function of glucosylceramide synthase. These mice will be used to interrogate whether ceramides, glucosylceramides, or both have a causative role in the pathogenesis of obesity and insulin resistance. Moreover, we will promote degradation of ceramides within glial cells, which we hypothesize will exacerbate sphingolipid oversupply to hypothalamic neurons during obesity. Of particular interest in each mouse model, evaluating if and how sphingolipid reduction in POMC neurons can improve whole-body insulin sensitivity independently of weight change will be assessed. This work will thus demonstrate a possible novel drug targets to decrease both body weight gain and blood glucose in obese or diabetic individuals, respectively. SA 1) Examine whether ceramide or glucosylceramide overaccumulation in POMC neurons contributes to high fat diet-induced obesity, insulin resistance, and impaired glucose tolerance. In order to determine the importance of abnormal sphingolipid accumulation in POMC neurons as mediators of diet-induced weight gain and insulin resistance we will force sphingolipid degradation (which lowers ceramide and glucosylceramides) or prevent glucosylceramide synthesis. SA 2) Determine if acid ceramidase overexpression in glial cells opposes high fat diet-induced obesity, insulin resistance, and impaired glucose tolerance. Novel tetracycline-inducible transgenic mice have been generated which overexpress acid ceramidase in glial cells. SA3) Examine whether sphingolipid overaccumulation in AgRP neurons contributes to high fat diet-induced obesity, insulin resistance, and impaired glucose tolerance. We will use tet-on transgenic lines overexpressing the ceramide degrading enzymes, acid ceramidase, to decrease AgRP sphingolipid. AgRP-specific glucosylceramide synthase knockout mice will be evaluated for their propensity for weight gain and impaired glycemic control. We will evaluate the importance of neuronal sphingolipids to both the initiation and reversal of obesity, insulin resistance, and glucose intolerance in each of these inducible mouse models.